The development of microfluidic technologies by the inventors and their co-workers has provided a fundamental paradigm shift in how artificial biological and chemical processes are performed. In particular, the inventors and their co-workers have provided microfluidic systems which dramatically increase throughput for biological and chemical methods, as well as greatly reducing reagent costs for the methods. In these microfluidic systems, small volumes of fluid (e.g., on the order of a few nanoliters to a few microliters) are moved through microchannels (e.g., in glass or polymer microfluidic devices).by electrokinetic or pressure-based mechanisms. Fluids can be mixed, and the results of the mixing experiments determined by monitoring a detectable signal from products of the mixing experiments.
Complete integrated systems with fluid handling, signal detection, sample storage and sample accessing are available. For example, Parce et al. xe2x80x9cHigh Throughput Screening Assay Systems in Microscale Fluidic Devicesxe2x80x9d WO 98/00231 and Knapp et al. xe2x80x9cClosed Loop Biochemical Analyzersxe2x80x9d (WO 98/45481; PCT/US98/06723) provide pioneering technology for the integration of microfluidics and sample selection and manipulation. For example, in WO 98/45481, microfluidic apparatus, methods and integrated systems are provided for performing a large number of iterative, successive, or parallel fluid manipulations. For example, integrated sequencing systems, apparatus and methods are provided for sequencing nucleic acids (as well as for many other fluidic operations, e.g., those benefiting from automation of iterative fluid manipulation). This ability to iteratively sequence a large nucleic acid (or a large number of nucleic acids) provides for increased rates of sequencing, as well as lower sequencing reagent costs. Applications to compound screening, enzyme kinetic determination, nucleic acid hybridization kinetics and many other processes are also described by Knapp et al.
As an alternative to microfluidic approaches, small scale array based technologies can also increase throughput of screening, sequencing, and other chemical and biological methods, providing robust chemistries for a variety of screening, sequencing and other applications. Fixed solid-phase arrays of nucleic acids, proteins, and other chemicals have been developed by a number of investigators. For example, U.S. Pat. No. 5,202,231, to Drmanac et al. and, e.g., in Drmanac et al. (1989) Genomics 4:114-128 describe sequencing by hybridization to arrays of oligonucleotides. Many other applications of array-based technologies are commercially available from e.g., Affymetrix, Inc. (Santa Clara, Calif.), Hyseq Technologies, Inc. (Sunnyvale, Calif.). and others. Example applications of array technologies are described e.g., in Fodor (1997) xe2x80x9cGenes, Chips and the Human Genomexe2x80x9d FASEB Journal. 11:121-121; Fodor (1997) xe2x80x9cMassively Parallel Genomicsxe2x80x9d Science. 7:393-395; Chee et al. (1996) xe2x80x9cAccessing Genetic Information with High-Density DNA raysxe2x80x9d Science 274:610-614; and Drmanac et al. (1998) xe2x80x9cAccurate sequencing by bridization for DNA diagnostics and individual genomics.xe2x80x9d Nature Biotechnology 16: 54-58.
The present invention is a pioneering invention in the field of microfluidics and mobile array technologies, coupling the fluid handling capabilities of microfluidic systems with the robust chemistries available through array technologies (e.g., solid phase chemistries) to facilitate laboratory and industrial processes. Many applications and variations will be apparent upon complete review of this disclosure.
The present invention provides microfluidic arrays. The arrays include particle sets (or xe2x80x9cpacketsxe2x80x9d) which can be mobile or fixed in position, e.g., within a microfluidic system. The particle sets can include fixed chemical components or can be modifiable. The arrays are used in a wide variety of assays, as chemical synthesis machines, as nucleic acid or polypeptide sequencing devices, as affinity purification devices, as calibration and marker devices, as molecular capture devices, as molecular switches and in a wide variety of other applications which will be apparent upon further review.
In one implementation, the invention provides microfluidic devices comprising one or more array(s) of particles. The device includes a body structure having a microscale cavity (e.g., microchannel, microchannel network, microwell, microreservoir or combination thereof) disposed within the body structure. Within the microscale cavity, an ordered array of a plurality of sets of particles (each particle set is constituted of similar or identical particle xe2x80x9cmembersxe2x80x9d or xe2x80x9ctypesxe2x80x9d) constitute the array. The array is optionally mobile (e.g., flowable in a microfluidic system, with flow being in either the same or in a different direction relative to fluid flow) or can be fixed (e.g., having flowable reagents flowed across the system).
The arrays of the invention include a plurality of particle sets. The precise location of the particle sets within the arrays is not critical, and can take many configurations. In one simple embodiment, particle sets abut in channels. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1,000, 10,000, 100,000 or more particle sets can abut in a single channel. Alternatively, non-abutting sets of particles dispersed within a microfluidic device can also be used, e.g., where the spatial location of each set of particles is known or can be determined. Fluidic reagents and particles are optionally flowed through the same or through different microchannels (or other microfluidic structures such as wells or chambers). For example, fluidic reagents are optionally flowed from a first channel into a second channel which includes particle sets of the array.
Particles (alternatively xe2x80x9cmicroparticlesxe2x80x9d) of the arrays of the invention can be essentially any discrete material which can be flowed through a microscale system. Example particles include beads and biological cells. For example, polymer beads, silica beads, ceramic beads, clay beads, glass beads, magnetic beads, metallic beads, inorganic beads, and organic beads can be used. The particles can have essentially any shape, e.g., spherical, helical, irregular, spheroid, rod-shaped, cone-shaped, disk shaped, cubic, polyhedral or a combination thereof. Particles are optionally coupled to reagents, affinity matrix materials, or the like, e.g., nucleic acid synthesis reagents, peptide synthesis reagents, polymer synthesis reagents, nucleic acids, nucleotides, nucleobases, nucleosides, peptides, amino acids, monomers, cells, biological samples, synthetic molecules, or combinations thereof. Particles optionally serve many purposes within the arrays, including acting as blank particles, dummy particles, calibration particles, sample particles, reagent particles, test particles, and molecular capture particles, e.g., to capture a sample at low concentration. Additionally the particles are used to provide particle retention elements. Particles are sized to pass through selected microchannel regions (or other microscale elements). Accordingly, particles will range in size, depending on the application, e.g., from about 0.1 to about 500 microns in at least one cross-sectional dimension.
In one aspect, the microfluidic system comprises an intersection of at least two microchannels. At least one member of the particle array is transported within a first of the at least two channels to a point proximal to or within the channel intersection. At least one of the reagents is transported through a second of the at least two intersecting microchannels to a point proximal to or within the channel intersection. The at least one member of the particle array and the at least one reagent are contacted proximal to or within the channel intersection.
Methods of sequencing nucleic acids are provided. In the methods, a first set of particles comprising at least one set of nucleic acid templates is provided, e.g., in a first microfluidic channel. A train of reagents (i.e., an ordered or semi-ordered arrangement of fluidic reagents in a channel) comprising a plurality of sequencing reagents is flowed across the first set of particles, or the first set of particles is flowed through the reagent train, depending on the application. This results in contacting the at least one set of nucleic acid templates with the plurality of sequencing reagents. Signals resulting from exposure of the first set of particles to the reagent train are selected, thereby providing a portion of sequence of the nucleic acid template. For example, the reagent train can include a polymerase, ae sufurylase, an apyrase, an inorganic phosphate, ATP, a thermostable polymerase, luciferin, luciferase, an endonuclease, an exonuclease, Mg++, a molecular crowding agent, a buffer, a dNTP, a dNTP analog, a fluorescent nucleotide, a chain terminating nucleotide, a reversible chain terminating nucleotide, a phosphatase, a reducing agent, an intercalator, a salt, DTT, BSA, a detergent (e.g., triton(copyright) or tween(copyright)), chemicals to inhibit or enhance EO flow (e.g., polyacrylamide), or other sequencing reagent. One preferred use for the arrays of the invention is sequencing by xe2x80x9csynthesisxe2x80x9d or xe2x80x9cincorporation,xe2x80x9d e.g., pyrosequencing. For example, the reagent train or array optionally include reagents for sequencing nucleic acid templates by pyrosequencing. A variety of other sequencing approaches are described herein.
Steps in the methods herein can be performed repeatedly or reiteratively for chemistries such as sequencing that involve repetitive synthesis and/or analysis steps. As reagents are depleted e.g., in the reagent train noted above, the method further optionally includes flowing a second train of reagents comprising a plurality of sequencing reagents across the first set of particles, or flowing the first set of particles through the second reagent. Alternatively, reagents are flowed in excess for a period of time, after which the channel(s) are rinsed, e.g., with a buffer before flowing a second reagent.
To further avoid contamination between repetitions or steps, methods are provided for loading and unloading reagents from a microfluidic device using a pair of split reagent wells and a pair of split waste wells.
Integrated systems and methods for performing fluidic analysis of sample materials in a microfluidic system having a particle array are also provided. For example, an integrated microfluidic system is provided which has a microfluidic device with the particle array, a material transport system, and a fluidic interface in fluid communication with the particle array. The interface samples a plurality of materials from one or a plurality of sources of materials and introduces the materials into contact with the particle array.
In the integrated methods, a first material from the plurality of materials is sampled with the fluidic interface. The first material is introduced into contact with at least one member of the particle array, whereupon the first sample material and at least a first member of the particle array react. A reaction product of the first sample material and the particle array is then analyzed and a second material (which may be the same as or different than the first material) is selected, based upon the reaction product. The second material is contacted with the particle array, where the second material and at least a second member of the particle array react. A second reaction product of the second material and the particle array is analyzed, thereby providing a fluidic analysis of the first and second materials.
For example, in sequencing applications, the first material can include a first DNA sequencing template, a first sequencing primer, or a first sequencing reagent while the second material can include a second DNA template, a second sequencing primer, or a second sequencing reagent. The array, in this example, includes a first mixture of reagents having DNA sequencing reagents or DNA templates. The first reaction product includes products of a DNA sequencing reaction (e.g., primer extension, sequencing by incorporation, e.g., by the pyrosequencing reaction or the like). A second sequencing primer is selected for inclusion in the second mixture of reagents based upon the products of the DNA sequencing reaction. Optionally, a third material is selected based upon the results of the analysis of the second reaction product. The third material is optionally introduced into proximity with the array, whereupon the third material and the array react. As above, the third reaction product is analyzed. This process is optionally reiterated several times (e.g., easily 10 times or more, often 100-1,000 times or more). Indeed, the process can be repeated thousands of times in a single experiment, e.g., to sequence a long stretch of DNA, or the like. Ordinarily, the integrated system includes a computer for performing or assisting in selection of the second material. The integrated system can also include fluid handling elements, e.g., electrokinetic or pressure flow controllers.
Systems for optimizing or performing a desired chemical reaction are provided. The system includes a microfluidic device which includes a microscale cavity having a particle array disposed therein. The particle array includes a plurality of particle sets. The system includes an electrokinetic or pressure based fluid direction system for transporting a selected volume of a first reactant to the array, or for reconfiguring the position of the array or for reconfiguring the arrangement of array members, or for loading array members (e.g., constituting a plurality of array sets) into the microscale cavity. The system also includes a control system, e.g., including a computer, which instructs the fluid direction system to deliver a first selected volume of first reactant to the array, or for moving members of the array into proximity with the first reactant, where contacting or mixing of the first reactant and at least one member of the array produces a first chemical reaction product. The control system optionally directs a plurality of mixings of the first reactant and the array (e.g., by electrokinetic and/or pressure based manipulation of reagent or array member as described herein), wherein a reaction condition selected from: temperature, pH, and time is systematically varied in separate mixings reactions. The system typically includes a detection system for detecting the first chemical reaction product e.g., as set forth above and supra. Other optional elements include a temperature control element for controlling temperature of reaction of the first and second element, a source of acid, a source of base and a source of reactants, reagents, array members, or the like.
In one aspect, the system instructs the fluid direction system to contact a second selected volume of the first or a second reactant with the array. This contact produces a second chemical reaction product.
The particles of the arrays optionally include a tag and one or more of the particle modification reagents comprising an anti-tag ligand. A xe2x80x9ctagxe2x80x9d is a component that can be detected, directly or indirectly (e.g., by binding to a detectable element). Exemplar tag and anti-tag ligands include nucleic acids; nucleic acid binding molecules, amidin, biotin, avidin, streptavidin, antibodies, antibody ligands, carbohydrate molecules, carbohydrate molecule binding reagents, proteins, protein binding molecules, organic molecules, organic molecule binding reagents, receptors, receptor ligands, etc. The particle modification reagent can also include a functional domain, e.g., independently selected from those noted for the tag and tag ligand. For example, in one embodiment, the one or more particle modification reagent has a nucleic acid having a biotin or avidin attached thereto.
Wash buffers, heat application, or an electric pulse are optionally used to strip components from arrays, thereby changing the array members. New components can be added to the array members following such washing. For example one or more particle modification reagents can be removed from one or more of the particle sets following washing, to provide one or more stripped particle sets. At least one additional particle modification reagent can be flowed across the one or more stripped particle set, thereby producing an additional particle set.
Many additional aspects of the invention will be apparent upon review including uses of the devices and systems of the invention, methods of manufacture of the devices and systems of the invention, kits for practicing the methods of the invention and the like. For example, kits comprising any of the devices or systems set forth above, or elements thereof, in conjunction with packaging materials (e.g., containers, sealable plastic bags etc.) and instructions for using the devices, e.g., to practice the methods herein, are also contemplated. Methods of Manufacture and manufactured devices comprising arrays or array members are set forth in detail herein.